This application is a U.S. national phase filing under 35 U.S.C. xc2xa7371 of PCT/EP93/01711, Feb. 7, 1993, and claims priority from Federal Republic of Germany application P 42 22 289.3, Jul. 7, 1992.
The invention relates to process for instabilizing viral quasi-species-distributions avoiding resistance phenomena by replication of the nucleic acids of the viruses present in the quasi-species-distribution by an defective replication system. Subject matter of the present invention is also an agent for executing the process according to the invention as well as nucleic acid sequences obtainable by reaction of viral replication systems with nucleotides.
Illnesses caused by viruses have been found in the hitherto existing pharmacology to be extremely difficult for treating by therapy. Up to now used conventional antiviral treatment strategies are exclusively concentrated to substances and methods having the aim to specifically suppress the reproduction of a virus in order to stop the infection in this way. This can be attained by different action mechanisms. A suitable point of attack of the reproduction of the virus is the specific virus replication system or one of the components thereof. In such a way, the viral replication apparatus can be inhibited by antimetabolites as AZT (HIV, AIDS) or Acyclovir (HSV, herpes). Other strategies are related to virus-coded enzymes which are assuming functions in quite specific replication phases of the virus. For instance, the viral protease for cleavage of polypeptide-translation products or also active principles which, e.g., prevent the built-up of a virus or prevent the release of the virus genom in the cell after infections, are belonging to
The mentioned strategies have in common that they will cause after a relatively short period of time resistance phenomena, such that the treated viruses avoid a drug therapy. In this case, the period of time, until the resistance phenomena will occur, can vary strongly for different active principles and viruses and can last for days up to years.
M. Eigen has been successful, to explain the property of the rapid adaptability of the quasi-species-distribution [M.Eigen (1971), Naturwissenschaften 58, 465-523]. By a quasi-species is understood that the xe2x80x9cwild-typexe2x80x9d-genome does not consist of a defined sequence which have all viruses of one species, but of a stable distribution of sequences of which the most frequent specific sequence is corresponding to the wild-type. It is identical with the consensus sequence which is obtainable if at every position or sequence the most frequently occurring nucleotide, respectively, is used [D. A. Steinhauer and J. J. Holland (1987), Ann. Rev. Microbiol. 41, 409-433]. However, the majority of the sequences is not identical with the wild-type sequence. That means, that indeed the viral genome is macroscopically defined and determinable as consensus sequence, but it is microscopically present in the form of a mixture of many mutants which are permanently in competition with one another. This competition maintains the genetic information of the virus in a dynamic equilibrium of mutation and selection. The genetic information modified by mutation is always re-established by selection, in such a way that the infection chain is maintained [Domingo, E., Sabo, D., Taniguchi, T. and Weissmann, L. (1978), Cell 13, 735-744].
By using a quasi-species-distribution, a population of microorganisms can adapt extremely rapidly to different environmental conditions. If, for instance, the temperature conditions change in the environment of a virus population, it is possible that the existing wild-type is in no way the variant which is reproducing most successfully under the new circumstances. Another variant among the great spectrum of the quasi-species-distribution is perhaps more suitable to replicate under the changed temperature. Possibly, also only some additional mutants are needed for a better adapted variant starting from a variant of the quasi-species-distribution instead of a wild-type sequence. By this mechanism, the great flexibility will become understandable, by which the microorganisms can adapt to changed environmental conditions in a surprisingly short period of time.
This flexibility can be paid by a high portion of lethal or partially defect descendants in the virus population. As a matter of fact, only a fractional part of the released viruses is infective in normal virus populations. The viral population can not stand to an increase of the rate of misincorporation of the viral replication system, e.g., in the case when the rate of misincorporation surpasses the theoretical error threshold belonging to the replication system. The portion of defect descendants will become that high that the infection chain can no more be maintained for a long time by viruses which still are infective.
For two reasons, this strategy is specifically directed against the distribution of viruses, especially of RNA-viruses. The viruses contain in most casesxe2x80x94contrary to their hostsxe2x80x94RNA as a genetic material. In order to enable a reproduction of the viruses, the virus must code itself at least the most important parts of the replication apparatus, since it can not fall back on the constituents of the host. Due to the lack of complicated error correcting mechanisms, narrow limits are set to the replication accuracy by the physical/chemical nature or the base-pair. The rates of misincorporation are realized in the range of between 10xe2x88x923 to 10xe2x88x925 with RNA-viruses. Furthermore, the selection is limited with viral genomes. Host cells divide after doubling their genomes and defect chromosomes become immediately apparent by disadvantages to the cell, as far as functionally needed gene places are concerned. Therefore, virus genomes are accumulated in infected cells to great populations. Defect gene products on single genomes do not cause disadvantages to the defect genome itself, because all genomes are participating in the same way to the common production. There is also no prejudice for defect matrices within the replication, such that the propagation of error is practically not hindered in the beginning. Only with some few viruses, the infective strands are read out in the late infection phase to a matrix (master template) in the meaning of a rolling-circle-model and, therefore, the propagation of error is controlled. Defects in the genome of a virus become only apparent during the next infection and only then can be eliminated by selection.
Theoretical calculations support the findings that a distribution of quasi-species-information can be maintained only in a stable way during facultative periods of time as long as a certain error threshold of the replication apparatus is not exceeded. Computer simulations have been found to give definite correlations between a pattern of selection advantages, a rate of replication error and the stability of the quasi-species-distribution. The calculation predicts a diffluence of information and therefore, the termination of a quasi-species-distribution in the vicinity of the wild-type sequence, if this error threshold is exceeded.
The relationships described by Eigen clarifies the dilemma in which the classical search for active agents is involved by screening of the active materials. When antiviral materials are discovered which prevent the propagation of the virus population by reproduction (replication), a xe2x80x9cselection pressurexe2x80x9d is exerted to the viral quasi-species-distribution, in such a way, that resistant mutants are formed which are the basis for a new viral quasi-species-distribution. Certainly, some of this quasi-species will be infective again, such that the viral infection is maintained by a new quasi-species-distribution. Therefore, the common conceptions of today for the so called xe2x80x9cdrug-screeningxe2x80x9d are even dangerous because of the permanent danger that new, possibly even stronger pathogenic viruses, are induced by this undesired selection pressure.